Oncolytic adenoviruses with increased proportion of the 156r splicing isoform of the e1b protein

ABSTRACT

The invention relates to a recombinant adenovirus that has an oncolytic effect in a cancer cell. By modulating the level and type of splice isoforms of the E1B gene product, expressed from the E 1 B gene, the oncolytic activity of such viruses can be enhanced. The invention provides a recombinant adenovirus in which the proportion of the E1B-156R isoformis increased relative to wild-type levels. Such a recombinant adenovirus may selectivity replicate in cancer cells, thereby killing cancer cells whilst sparing normal cells.

TECHNICAL FIELD

This invention relates to oncolytic adenoviruses and their use intreating neoplastic disease. More particularly, the invention relates tomutations of the E1B gene that modulate the levels of splicing isoformsof this gene, thereby improving the therapeutic index of the oncolyticadenovirus.

BACKGROUND TO THE INVENTION

Oncolytic Viruses

Oncolytic virotherapy is an emerging treatment platform for cancertherapy. Oncolytic viruses are viruses that selectively replicate incancer cells that possess specific oncogenic phenotypes, thereby killingcancer cells whilst sparing normal cells. Initial research focussed onnaturally-occurring non-pathogenic viruses, however, these studies wereof limited success. Although tumour growth was observed to slow down andnormal tissue was not damaged, there was no alteration in the course ofthe disease.

Recent studies have therefore focussed on engineering recombinantviruses that selectively target cancer cells. One example of this classof engineered viruses is adenoviruses that are mutated in the E1B regionof the viral genome.

Adenoviral E1B and p53

One function of the mammalian tumour suppressor protein p53 is tomediate cell-cycle arrest and/or apoptosis in response to DNA damage orforeign DNA synthesis. Consequently, some viruses, such as adenovirus,encode proteins that inactivate p53 in infected cells to allow efficientviral replication. One of these proteins, the 55 kiloDalton protein fromthe E1B region of adenovirus (E1B-55K or E1B-496R), binds to p53 socausing a substantial loss of p53. This consequently preventsp53-mediated apoptosis of the infected cell. E1B-496R is thereforeessential for adenoviral replication in cells containing functional p53.

Human tumour cells are frequently homozygous or heterozygous for mutated(e.g. substitution, deletion, frameshift mutated) p53 alleles, and lackp53 function necessary for normal control of the cell cycle (Hollsteinet. al (1991) Science 253:49; Levine et al. (1991) Nature;351(6326):453-6). Many neoplastic cells are therefore p53⁽⁻⁾ eitherbecause they lack sufficient levels of p53 and/or because they expressmutant forms of p53 which are incapable of substantial p53 function.

E1B Mutated Adenoviruses

Oncolytic adenoviruses have been engineered that take advantage of thedifference in p53 functionality between neoplastic and normal cells. Bymutating the E1B-496R protein to remove binding interactions with p53 orby making various deletions within the E1B locus (see, for example, U.S.Pat. No. 5,677,178), the resulting adenoviruses can replicate andultimately lyse cancer cells that substantially lack p53 function, butnot in cells that possess normal p53 function.

One example is ONYX-015 (originally named d11520 and also referred to asH101), a mutant adenovirus that does not express the E1B-496R protein(Heise et al. (1997) Nat. Med. 3 (6): 639-645). The virus contains astop codon immediately following the translation start codon and alsohas a large deletion of the E1B-496R coding sequence. As a result thisvirus lacks the ability to bind and inactivate p53, and thus can onlyreplicate efficiently in cells defective in p53 function, such asneoplastic cells and tumours. Unfortunately, E1B-496R carries out otherfunctions in addition to binding and inactivating p53 (Eager et al.(2001) Cancer Gene Ther. 18 (5): 305-317). Consequently, the ONYX-015virus is defective in cytoplasmic accumulation of the viral late mRNAs,host cell shut-off and translation of late mRNAs. Thus, the mutation inONYX-015 compromises the ability of the mutant virus to reproduce itselfin tumour cells. An additional problem is that large deletionsdestabilise the viral genome.

Additional examples are ONYX-051 and ONYX-053, mutant adenoviruses thatcontain point mutations (R240A and H260A, respectively) in the E1B-496Rprotein that prevent its binding to p53. These mutations enable thevirus to replicate selectively in cells that are deficient in p53function, without compromising the ability of the virus to replicate inthese cells (Shen et al. (2001) J. Virol. 75 (9): 4297-4307 and U.S.Pat. No. 7,785,887).

However, there remains a great need for improved mutant viruses whoseoncolytic ability has been enhanced and which are useful in the therapyof cancer.

DISCLOSURE OF INVENTION

It has now been found by the present inventor that by modulating thelevel and type of splice isoforms of the E1B gene product, expressedfrom the E1B gene, the oncolytic activity of such viruses can beenhanced. Accordingly, a first aspect of the present invention providesa recombinant adenovirus in which the proportion of the E1B-156R isoformis increased relative to wild-type levels, wherein the adenovirus has anoncolytic effect in a cancer cell. The recombinant adenovirus may carrya mutation such that the proportion of the E1B-156R isoform is increasedrelative to wild-type levels, so that the adenovirus has an oncolyticeffect in a cancer cell. The mutation may be in the sequence of the E1Bgene of the adenovirus. A virus according to the invention is thereforereplication-inhibited in non-neoplastic cells but is capable ofexpressing a replication phenotype in neoplastic cells, includingneoplastic cells that substantially lack functional p53.

In the specific examples of adenoviruses described herein,over-expression of E1B-156R is thought to be an imbalance caused by amutated 93R splice site in the E1B gene of the adenovirus. 156R is ableto complement some of the 496R function, but not the ones essential tooncoselectivity. In contrast to prior art viruses of similar type,viruses according to the present invention include a functional E3Bregion for better in vivo efficacy. For example, Onyx-015 lacks E3B.Indeed, Onyx-015 virus and its selectivity is by far outperformed byviruses according to the invention, in all respects. Furthermore, theinventor has tested viruses prepared in accordance with the invention innormal cells and the results show that the viruses have an outstandingsafety profile, especially in comparison to the known virus Onyx-015.

Herein, the term “replication-inhibited virus” or“replication-defective” refers to a virus that preferentially inhibitscell proliferation or induces apoptosis in a predetermined cellpopulation that is transformed into a cancerous or neoplastic state.Such a virus is substantially unable to inhibit cell proliferation,induce apoptosis, or express a replication phenotype in cells comprisingnormal p53 function levels that are characteristic of non-replicating,non-transformed cells. Such transformed cells may substantially lack p53function, which supports expression of a virus replication phenotype.However, selectivity of viruses according to the invention forneoplastic tissue might well be more general than just for p53 status;the transformed state as such might be the basis for selection. Forexample, it has been suggested that oncolytic selectivity observed withthe ONYX-015 virus may be due to the capacity of some cancer cell linesto support late viral RNA export from the nucleus, a function which islost in ONYX-015 in normal cells due to the E1B-496R deletion. A similarmechanism may operate in the recombinant adenoviruses of the presentinvention, which have reduced levels of E1B-496R protein. It is not asyet clear exactly how an increase in the levels of E1B-156R in therecombinant adenoviruses of the present invention results in oncolyticselectivity.

Typically, a replication-inhibited virus according to the inventionexhibits a substantial decrease in plaquing efficiency on cellscomprising normal p53 function (for a suitable assay, see Wang, Y., G.Hallden, et al. (2003). “E3 gene manipulations affect oncolyticadenovirus activity in immunocompetent tumor models.” Naturebiotechnology 21(11): 1328-1335). Another example of a suitable assaythat may be used is a cytotoxicity assay to measure loss of viablecells, using for example a tetrazolium dye such as MTT, XTT, MTS or aWST (see Berridge et al., Biotechnology Annual Review, 11: 127-152(2005).

As used herein, the term “replication phenotype” refers to one or moreof the following phenotypic characteristics of cells infected with avirus such as a replication-inhibited adenovirus: (1) substantialexpression of late gene products, such as capsid proteins (e.g.,adenoviral penton base polypeptide) or RNA transcripts initiated fromviral late gene promoter(s); (2) replication of viral genomes orformation of replicative intermediates; (3) assembly of viral capsids orpackaged virion particles; (4) appearance of cytopathic effect (CPE) inthe infected cell; (5) completion of a viral lytic cycle; and (6) otherphenotypic alterations which are typically contingent upon abrogation ofp53 function in non-neoplastic cells infected with a wild-typereplication-competent DNA virus encoding functional oncoprotein(s). Areplication phenotype according to the present invention comprises atleast one of the phenotypic characteristics listed above, preferablymore than one of the phenotypic characteristics, such as 2, 3, 4, 5, 6or more characteristics.

Techniques for the measurement of these phenotypes will be known tothose of skill in the art. For example, methods to assess appearance ofCPE are described in the examples herein, and evaluated using 50% tissueculture infective dose (TCID₅₀) and number of plaque-forming units(pfu)/cell (cell count on the day of infection).

The term “neoplastic cells” refers to cells which exhibit relativelyautonomous growth, so that they exhibit an aberrant growth phenotypecharacterised by a significant loss of control of cell proliferation.Neoplastic cells comprise cells which may be actively replicating or ina temporary non-replicative resting state (G1 or GO); similarly,neoplastic cells may comprise cells that have a well-differentiatedphenotype, a poorly differentiated phenotype, or a mixture of both typesof cells. Thus, not all neoplastic cells are necessarily replicatingcells at a given time point. The set of cells defined herein asneoplastic cells consists of cells in benign neoplasms and cells inmalignant (or frank) neoplasms. Frankly neoplastic cells are frequentlyreferred to as cancerous, typically carcinoma if originating from cellsof endodermal or ectodermal histological origin, or sarcoma iforiginating from cells types derived from mesoderm. The terms neoplasticcell and cancer cell are used interchangeably herein.

Herein, the term “p53 function” refers to the property of having anessentially normal level of a polypeptide encoded by the p53 gene (i.e.relative to non-neoplastic cells of the same histological type), whereinthe p53 polypeptide is capable of binding to wild-type adenovirusE1B-496R polypeptide. For example, p53 function may be lost byproduction of an inactive (i.e. mutant) form of p53 or by substantialdecrease or total loss of expression of p53 polypeptide. p53 functionmay also be substantially absent in neoplastic cells that comprise p53alleles that encode wild-type polypeptide; for example, a geneticalteration outside of the p53 locus, such as mutations that result inaberrant subcellular processing or localisation of p53 (e.g. a mutationresulting in localisation of p53 predominantly in the cytoplasm ratherthan the nucleus) can result in loss of p53 function. Many neoplasticcells are therefore p53⁽⁻⁾ either because they lack sufficient levels ofp53 and/or because they express mutant forms of p53 which are incapableof substantial p53 function. In the context to the present invention,the key function of p53 is the ability to mediate cell-cycle arrestand/or mediate apoptosis in response to DNA damage or foreign DNAsynthesis. Neoplastic cells lack functional p53, if the said reductionin p53 function prevents normal control of the cell cycle and apoptosis.This may consist a decrease of 2-fold, 5-fold, 10-fold, 20-fold,50-fold, 100-fold or more of correctly processed, localised andexpressed p53 that can bind to E1B-496R, compared to the correspondingnon-neoplastic cells of the same type. These cells are therefore termed“p53^((−)n)”.

It is believed that replication-deficient adenovirus species which lackthe capacity to complex p53 but substantially retain other essentialviral replicative functions will exhibit a replication phenotype incells which are deficient in p53 function (e.g., cells which arehomozygous for substantially deleted p53 alleles, or cells whichcomprise mutant p53 proteins which are essentially non-functional) butwill not substantially exhibit a replicative phenotype innon-replicating, non-neoplastic cells. Such replication inhibitedadenovirus species are referred to herein for convenience as E1B-p53⁽⁻⁾replication-deficient adenoviruses.

An “oncolytic virus” is a virus that preferentially infects and lysescancer cells. Oncolytic effect is seen when comparing efficacy in cancercells versus normal cells. A virus is considered oncolytic if the ratioof lysed cancer cells to non-cancerous cells is 2:1, 4:1, 10:1, 20:1,50:1, 100:1 or more. Preferably, a virus is identified as oncolytic byassessing the oncolytic index (see below). Herein, the oncolytic effectcomprises a) viral infection of cells; b) selective replication of theviral genome in (p53 function-deficient) cancer cells leading topreferential virus-mediated cell lysis in cancer cells (which may be p53deficient), and the release of viral particles for further infectionevents. The oncolytic effect can be measured using various assays. Inthese assays a control virus should be used, often the wildtype/naturally occurring version. As such any of the following examplesof assays can be used: MTS (cytotoxicity), TCID₅₀ (replicationcompetency), LDH assays (lactate dehydrogenase (LDH) is a stable enzyme,present in all cell types, and is rapidly released into the cell culturemedium upon damage of the plasma membrane), FACS (cell sorting), westernblot, and QPCR (late gene expression or genome copy number). Otherexamples will be clear to those of skill in the art.

An advantage of a virus according to the present invention is that sucha virus has minimal genetic aberrations. Preferably, a virus accordingto the invention will be mutated in its genetic sequence, such as in theform of point mutations (including insertions, deletions, additions, andsubstitutions); point mutations are better for the health of the virus.Larger changes put an evolutionary strain on the virus. Additionally,genomic size and integrity can be important.

A virus according to the present invention has a cancer selection indexor oncolytic index (the two terms are interchangeable) that is muchimproved in comparison to existing oncolytic viruses that are available,such as the H101 virus. This refers to the replication capacity innormal cells in comparison to cancer cells, which may be expressedaccording to the following equation:

(OVc/OVn)/(WTc/WTn)=Cancer selection index

wherein OV=Oncolytic virus replication capacity; WT is Control virusreplication capacity; c is cancer cells; n is normal cells.

A virus according to the invention may have a cancer selection index ofbetween 2 and 10,000, depending on the type of cell, preferably between5 and 5000, 10 and 1000, or 50 and 500. In the case of a representativeexample of a virus according to the invention, Ixovex, this virusexhibits a cancer selection index versus control of 3.5 in HeLa cells;14 in A549 cells; 2000 in H1299 cells and 450 in H460 cells. In the samecell types, the values for the Onyx-015 virus versus Ixo-ctrl areHeLa=0.1x; A549=0.1x; H1299=0.05x; and H460=0.004x.

As such, it is possible to apply a virus according to the invention tothe majority of tumour types. One theory is that the oncolytic virusesof the invention are selective for p53 negative status andquickly-replicating cells.

In addition, a virus according to the invention will be met by the hostimmune defence and ultimately cleared, this before the complete tumoureradication. This does not only present itself as a way of removing thevirus, so negating any possible liver toxicity as a result of a viraloverload, but also provides the chance to induce an anticancer immuneresponse in the host since the immune system will be alerted to theviral presence in the tumour.

Adenoviruses

A virus according to the invention is a recombinant adenovirus. At thetime of writing, there are more than 65 described serotypes in humans(HAdV-1 to 65) distributed across seven species (Human adenovirus A toG) and as many from other mammals and birds (see Strauss, “Adenovirusinfections in humans,” in The Adenoviruses (1984) ed. Ginsberg, pp.451-596 Plenum Press, New York. For a general description of adenovirusbiology see Virology, Second Edition, eds. Fields and Knipe. Vol. 2, pp1651-1740, Raven Press, New York). The term “adenovirus” as used herein,encompasses any one of these adenovirus species. Preferably, anadenovirus according to the invention is a human adenovirus of subfamilygroup C, namely one of serotypes 1, 2, 5, 6, or 57. More preferably, theterm adenovirus applies to two human serotypes, Ad2 and Ad5.

In one preferred embodiment of the invention, the adenovirus isadenovirus serotype Ad5. The adenovirus may be adenovirus serotype Ad5strain pTG3602. Strain pTG3602 has approximately 15 point mutationsscattered throughout the 35,000 nucleotide adenovirus genome, howevernone of these mutations fall within the E1B gene. Herein, adenovirustype 5 provides a common reference point for the nucleotide numberingconvention of viral polynucleotides and amino acid numbering ofviral-encoded polypeptides the E1B viral gene region. Those skilled inthe art will readily identify the corresponding positions in otheradenoviral serotypes. Herein, the term “recombinant” indicates that apolynucleotide construct (e.g. and adenovirus genome) has beengenerated, in part, by intentional modification by man.

E1B Gene

A virus according to the invention preferably carries a mutation in thesequence of the E1B gene. All serotypes encode a gene that is referredto across serotypes as early region 1B (E1B), encoding gene products ofthe early phase of DNA replication. Herein, the “E1B gene” refers to thefull length transcription unit of the E1B gene in any adenovirus,preferably human adenovirus. A representative example of an E1B gene isthat from adenovirus type 5 (Ad5) which has the polynucleotide sequenceaccording to SEQ ID NO: 1. Other examples will be known to the skilledreader and details can be found in commonly used databases, such as, forexample Entrez Gene (http://www.ncbi.nlm.nih.gov/gene). In humanadenovirus type 5, the E1B coding region starts at genomic nucleotidenumber 1714 and ends at the E1B polyA site at genomic nucleotide number4043. Similar regions are present in all adenoviruses so far tested, forexample, including species as diverse as sheep, snake and even batadenovirus.

The E1B transcription unit of the human adenovirus encodes at least fivedifferent splicing isoforms (see FIG. 2) (Takayesu et al. (1994) J. Gen.Vivol. 75:789-798). Again, giving the example of Ad5, the major 2.28 kbE1B precursor mRNA encodes two overlapping reading frames, one for the176 residue E1B-19K protein (E1B-176R) and the other for E1B-55K protein(496 residues; E1B-496R). The E1B-156R, E1B-93R, E1B-84R isoforms (namedafter the number of amino acids in the expressed product) are generatedby alternative splicing of the precursor mRNA for E1B-496R, between acommon splice donor (SD1) and one of three splice acceptor sites(SA1-3). The resulting mRNAs encode the 79 amino acids of the E1B-496RN-terminus, and whilst E1B-93R and E1B-84R have unique C-termini,E1B-156R is completed by the 77 C-terminal residues of E1B-496R.Alternative splicing is explained in Kelemen, O., P. Convertini, et al.(2012). “Function of alternative splicing.” Gene. It will be apparent tothe skilled person that the E1B isoforms in other adenovirus serotypesmay have slightly different lengths to those discussed above for Ad5(e.g. the equivalent of E1B-156R in Ad2 is 155 amino acids long and istherefore often referred to as 155R). Herein, the isoform namesE1B-156R, E1B-93R, E1B-84R, E1B-176R and E1B-496R refer to theequivalent isoform of the same approximate size in all adenoviruses,regardless of the actual number of amino acids in the equivalentisoform.

It has been confirmed that the E1B-156R isoform exists in a widecross-section of adenovirus variants, by using PCR to amplify thespecific cDNAs for E1B-156R using start and stop primers specific foreach respective E1B-55k gene (FIG. 14). Our experiments show similarsplicing patterns in representative viruses from each of the differentgenera (A-Ad12, B1-Ad3, B2-Ad11, C-Ad5, D-Ad37, E-Ad4 and F-Ad40).Indeed, Ad1wt and Ad57wt have identical E1B-156R protein sequences;Ad2wt and Ad6wt also have identical sequences; and Ad5wt differs onlyslightly from them all. This makes only three different E1B-156R proteinsequences in the entire subfamily C differing at a total of five singleamino acid positions and in the length of an internal poly-alaninestretch. Thus it is fully expected that the results demonstrated hereinin serotypes Ad2 and Ad5 will be mirrored across other adenovirusvariants.

A number of complementation experiments have been performed to show thatan increase in E1B-156R is responsible (at least in part) for theincrease in Oncolytic Index (OI) that has been observed, such thatoverexpression of the Ad5-156R gives a potent increase in the OI ofadenoviruses generally. In FIGS. 11A, B and C and FIG. 12 herein, it isshown that adenovirus type 5 E1B-156R is a potent enhancer of the OI inthe subfamily group C. Furthermore, the E1B-156R equivalent from Ad2wtwas shown to have a positive effect on the OI of Ad5wt, meaning that theeffect appears not to be limited to one particular adenovirus serotype.

Herein, the E1B-156R isoform of human Ad5 has the polynucleotidesequence according to SEQ ID NO: 2 and the polypeptide sequenceaccording to SEQ ID NO: 3. Herein, the 496R isoform has thepolynucleotide sequence according to SEQ ID NO: 4 and a polypeptidesequence according to SEQ ID NO: 5. Herein, the E1B-93R isoform has thepolynucleotide sequence according to SEQ ID NO: 6. and the polypeptidesequence according to SEQ ID NO: 7. Herein, the E1B-84R isoform has thepolynucleotide sequence according to SEQ ID NO: 8 and the polypeptidesequence according to SEQ ID NO: 9. It will be appreciated by theskilled reader that a degree of variation in sequence exists innaturally-occurring viral variants; accordingly, the invention embracesisoform sequences that differ from the specific sequences set out in thereference sequences referred to about, but are 80%, 85%, 90%, 95%, 98%,99% or more homologous or identical to those sequences, as calculated bycommon sequence alignment programs, for example, BLAST(http://blast.ncbi.nlm.nih.gov/Blast.cgi) which can be nucleotide BLAST(blastn) or protein BLAST (blastp). Two sequences are said to be“homologous”, as the term is used herein, if one of the sequences has ahigh enough degree of identity or similarity to the other sequence.“Identity” indicates that at any particular position in the alignedsequences, the nucleotide is identical between the sequences.“Similarity” indicates that, at any particular position in alignedpolypeptide sequences, the amino acid residue is of a similar typebetween the sequences. Degrees of identity and similarity can be readilycalculated (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing. Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part 1, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991).

Accordingly, embodiments of the invention include variant recombinantadenoviruses where the E1B-156R isoform has a polynucleotide sequencethat has at least 80% sequence identity to SEQ ID NO: 2 and apolypeptide sequence that has at least 80% sequence identity to SEQ IDNO: 3; where the E1B-496R isoform has a polynucleotide sequence that hasat least 80% sequence identity to SEQ ID NO: 4 and a polypeptidesequence that has at least 80% sequence identity to SEQ ID NO: 5; wherethe E1B-93R isoform has a polynucleotide sequence that has at least 80%sequence identity to SEQ ID NO: 6 and a polypeptide sequence that has atleast 80% sequence identity to SEQ ID NO: 7; and where the E1B-84Risoform has a polynucleotide sequence that has at least 80% sequenceidentity to SEQ ID NO: 8 and a polypeptide sequence that has at least80% sequence identity to SEQ ID NO: 9. Representative examples ofvariant E1B-156R isoform sequences are given herein (FIG. 13). Includedequivalent sequences to Ad5 E1B-156R are those with 80%, 85%, 90%, 95%,98%, 99% or more identity with those sequences provided in FIG. 13 (asdescribed above for Ad5). Modulation of isoform levels by engineering.

According to the invention, the level and/or type of splice isoforms ofthe E1B gene product, expressed from the E1B gene, is modified, and as aresult the viruses are rendered oncolytic. The levels and/or types ofE1B isoforms may be modulated by any means, for example using ribozymesdesigned to specifically cleave the E1B isoform mRNAs at selectedpositions and thereby preventing translation of the mRNAs intofunctional polypeptide. Alternative methods will be apparent to those ofskill in the art, and include insertion of multiple copies of the E1Bgene sequence, or forms that encode E1B-156R; activation of regulationof E1B gene expression, or forms that encode E1B-156R, for example bymodulation of promoter or enhancer sequences; insertion of regulatorysequences, and so on.

Herein, we provide as particular examples of variant adenovirusesaccording to the invention, adenoviruses that include one or moremutations in the splicing regions of E1B gene that achieve this effect.As splice site recognition by the spliceosome is known to be affected bymRNA secondary structure, mutations in the E1B gene that affect thesecondary structure of its mRNA may also modulate the levels and typesof E1B isoforms. For example, the mutation may remove a splice site bychanging the polynucleotide and polypeptide sequence of the E1B gene; ormay remove a splice site by changing the polynucleotide sequence of theE1B gene and retaining the original polypeptide sequence.

In one embodiment, this effect may be achieved when the E1B gene ismutated in one or more of the splicing recognition regions comprising:a) the splice donor site 1 (SD1); the E1B-93R splice acceptor (SA1); c)E1B-156R splice acceptor (SA2); d) E1B-84R splice acceptor (SA3); and/ore) splice donor site 2 (SD2).

In the example case of Ad5, these splice sites are at the followingpositions and have the following sequences: SD1 has the sequence GTGGCat position 2251-2255 of the Ad5 genome (position 2256-2260 of the Ad5genome accession number AC_(—)000008.1 (SEQ ID NO: 41) and position543-547 in the E1B gene (SEQ ID NO: 1)). The E1B-93R splice acceptor(SA1) has the sequence AACAG at position 3218-3222 of the Ad5 genome(position 3213-3217 of the Ad5 genome accession number AC_(—)000008.1(SEQ ID NO: 41) and position 1500-1504 in the E1B gene (SEQ ID NO: 1)).The E1B-156R splice acceptor (SA2) has the sequence TTGAG at position3276-3280 of the Ad5 genome (position 3271-3275 of the Ad5 genomeaccession number AC_(—)000008.1 (SEQ ID NO: 41) and position 1558-1562in the E1B gene (SEQ ID NO: 1)). The E1B-84R splice acceptor (SA3) hasthe sequence TGCAG at position 3595-3599 of the Ad5 genome (position3590-3594 of the Ad5 genome accession number AC_(—)000008.1 (SEQ ID NO:41) and position 1877-1881 in the E1B gene (SEQ ID NO: 1)). The splicedonor site 2 (SD2) has the sequence GTACT at position 3506-3510 of theAd5 genome (position 3511-3515 of the Ad5 genome accession numberAC_(—)000008.1 (SEQ ID NO: 41) and position 1798-1802 in the E1B gene(SEQ ID NO: 1)). Equivalent sites at equivalent positions in other humanserotypes will be easily apparent to those of skill in the art, imbuedwith the teaching of the present invention.

Accordingly, one aspect of the present invention is a recombinantadenovirus in which where the E1B gene splicing recognition regions aremutated at one or more of the following positions in the Ad5 genome: a)nucleotide 3216 of the adenovirus Ad5 genome (accession numberAC_(—)000008.1) (SEQ ID NO: 41) (position 1503 in the E1B gene (SEQ IDNO: 1); b) nucleotide 3218 of the adenovirus Ad5 genome (accessionnumber AC_(—)000008.1) (SEQ ID NO: 41) (position 1505 in the E1B gene(SEQ ID NO: 1); and/or c) nucleotide 3221 of the adenovirus Ad5 genome(accession number AC_(—)000008.1) (SEQ ID NO: 41) (position 1508 in theE1B gene (SEQ ID NO: 1). The E1B gene may contain one or more of thefollowing mutations: a) A3216G in the adenovirus Ad5 genome (position1503 in the E1B gene (SEQ ID NO: 1)); b) G3218A in the adenovirus Ad5genome (position 1505 in the E1B gene (SEQ ID NO: 1)); and/or c) G3221Ain the adenovirus Ad5 genome (position 1508 in the E1B gene (SEQ ID NO:1)). Herein, the positions of all point mutations are numbered accordingto Ad5 genome accession number AC_(—)000008.1 (SEQ ID NO: 41).

The table below identifies the sequences and positions of the splicingrecognition regions of the E1B gene in the Ad5 genome. “Ad 5 genomeposition” and “E1B gene position” correspond to the five residuesimmediately upstream of splice donor sites (SD1 and SD2), andimmediately downstream of splice acceptor sites (SA1, SA2 and SA3).

E1B Ad5 E1B  splice genome gene SEQ ID sites position position SequenceNO: splice 2251- 538-542 cag/ GTGGC TGAAC SEQ ID donor 1 2255 NO: 10(SDI) E1B- 3218- 1505- TCCTTGCATTTGGG SEQ ID 93R 3222 1509 T AACAG /gagNO: 11 splice acceptor (SA1) E1B- 3276- 1563- ACACTAAGATATTG SEQ ID 156R3280 1567 C TTGAG /ccc NO: 12 splice acceptor (SA2) E1B- 3595- 1882-GTCTTATGTAGTTTTGTAT SEQ ID 84R 3599 1886 CTGTTT TGCAG /cag NO: 13 spliceacceptor (SA3) splice  3506- 1793- gag/GTACTGAAAT SEQ ID  donor 2 35101797 NO: 14 (SD2)

The “forward slash” indicates the actual splice site.

Mutation of the viral sequence within the splicing recognition regionsof the E1B gene can involve either a) removing a splice site by changingthe polynucleotide and polypeptide sequence of the E1B gene; or b)removing a splice site by changing the polynucleotide sequence of theE1B gene and retaining the original polypeptide sequence. As the skilledperson will appreciate, there is redundancy in the genetic code, i.e.some amino acids are encoded by multiple codons. The splice sitesequences can be removed from the transcribed E1B mRNA by mutating thecorresponding adenoviral DNA to use (an) alternative codon(s) for theamino acids the polynucleotide sequence is encoding at these sites.Thus, the resulting translated protein will preferably not contain anyamino acid changes. The codon table below shows the redundancy in thegenetic code.

Amino Acids Codons Alanine Ala A GCA GCC GCG GCU Arginine Arg RAGA AGG CGA CGC CGG CGU Aspartic acid Asp D GAC GAU Asparagine Asn NAAC AAU Cysteine Cys C UGC UGU Glutamic acid Glu E GAA GAG Glutamine GlnQ CAA CAG Glycine Gly G GGA GGC GGG GGU Histidine His H CAC CAUIsoleucine Ile I AUA AUC AUU Leucine Leu L CUA CUC CUG CUU UUA UUGLysine Lys K AAA AAG Methionine Met M AUG Phenylalanine Phe F UUC UUUProline Pro P CCA CCC CCG CCU Serine Ser S AGC AGU UCA UCC UCG UCUThreonine Thr T ACA ACC ACG ACU Tryptophan Trp W UGG Tyrosine Tyr YUAC UAU Valine Val V GUA GUC GUG GUU Stop TAA TGA TAG

In a preferred embodiment, increased levels of the E1B-156R isoform areachieved as a result of a mutated 93R splice site in the E1B gene.

In a preferred embodiment, the E1B gene splicing recognition regions aremutated at one or more of the following positions: a) nucleotide 3216(e.g. cagGAGG→cggGAGG) of the adenovirus Ad5 genome (accession numberAC_(—)000008.1) (SEQ ID NO: 41); b) nucleotide 3218 of the adenovirusAd5 genome (accession number AC_(—)000008.1) (SEQ ID NO: 41); and/or c)nucleotide 3221 of the adenovirus Ad5 genome (accession numberAC_(—)000008.1) (SEQ ID NO: 41). Equivalent mutations in otheradenovirus serotypes will be clear to the skilled reader.

In a more preferred embodiment, the E1B gene contains one or more of thefollowing mutations: a) A3216G (cagGAGG→cggGAGG); b) G3218A; and/or c)G3221A, corresponding to positions 1503, 1505 and 1508 in the E1B gene(SEQ ID NO: 1) respectively. Equivalent mutations in other adenovirusserotypes will be clear to the skilled reader.

Levels of Isoforms

Any mutation that is introduced into the sequence of an adenovirusgenome should have the effect that the proportion of at least one of theE1B splicing isoforms, E1B-156R, E1B-496R, E1B-93R, and E1B-84R, (andpotentially two, three, or all four isoforms) varies with respect tolevels that are present in the wild-type under similar conditions.Preferably, the proportion of the E1B-496R isoform is decreased relativeto wild-type levels, or even totally shut down. Alternatively, theproportion of the E1B-156R isoform is increased relative to the E1B-496Risoform, the proportion of the E1B-156R isoform is increased relative tothe E1B-93R isoform and/or the proportion of the E1B-156R isoform isincreased relative to the E1B-84R isoform. These changes in levels ofparticular isoforms have the effect of enhancing oncolysis, which canalso be expressed as enhancing the oncolytic index.

The level of the E1B-156R isoform may be increased relative to theE1B-156R in the equivalent wild type adenovirus sequence. Herein,“increased” means that the proportion of the E1B-156R isoform isincreased at least 2-fold, 4-fold, 10-fold, 20-fold, 50-fold, 100-fold,1,000-fold or 10,000-fold relative to wild type levels.

Herein, “decreased” means that the proportion of the E1B-496R isoform isdecreased at least 2-fold, 4-fold, 10-fold, 20-fold, 50-fold, 100-fold,1,000-fold or 10,000-fold relative to wild type levels.

Herein, “wild type levels” refers to the levels of E1B-156R, E1B-496R,E1B-93R, and E1B-84R isoforms that are evident by expression of theseproteins from the wild-type adenovirus, with no mutations in the E1Bgene reference sequence (i.e. SEQ ID NO: 1). For example, the proportionof the mutant Ad5 E1B-156R isoform may be increased relative to levelsin wild-type Ad5 adenovirus, such that mutant adenovirus has anoncolytic effect in a cancer cell. Mutant Ad2 E1B-156R isoform may beincreased relative to levels in wild-type Ad2 adenovirus.

Preferred viruses according to the invention may have diminished orinhibited expression of E1B-93R, and preferably do not express E1B-93R.By “does not express” is meant herein that the detectable level of theE1B-93R sequence is less than 50%, 10%, 1% of the level of the E1B-93Rsequence in the wild type adenovirus under equivalent conditions,preferably less than 0 1%, less than 0 01% or even less. This has theeffect of raising the expression of E1B-156R.

Preferably, an optimal ratio of the E1B-156R, E1B-496R, E1B-93R, andE1B-84R isoform protein levels would lie along the lines of about67:0:0:33 as compared to about 5:70:10:15 for wild-type viruses. As theskilled reader will appreciate, however, it is difficult or impossibleto be exact about relative levels of this type since they are dependenton the point in the infection cycle assessed, i.e.early/intermediate/late. The ratios changes for the benefit of theshorter spliceforms at the cost of 496R (which is the unspliced,full-length RNA). In particular, favoured ratios of the E1B-156R isoformto the E1B-496R isoform are 2:1, 5:1, 10:1, 20:1, 50:1, 100:1, 1000:1 or10,000:1 or more. A ratio of 100:1 or more is preferred.

Herein reference to “the proportion” of the E1B-156R, E1B-496R, E1B-93R,or E1B-84R isoforms refers to: a) the level of the isoform protein thatis expressed; and/or b) the level of the isoform mRNA that is produced.

Techniques for measuring mRNA levels will be known to those of skill inthe art for the quantitation of polynucleotides, such as, for example,nucleic acid amplification, for instance PCR, RT-PCR, TaqMan-basedmethodologies, RNase protection, Northern blotting and in situhybridization techniques, and quantitative versions of these methods.Changes in mRNA expression levels may be of a temporal, spatial orquantitative nature. The number of copies of each E1B isoform mRNA canbe calculated and compared to a reference, for example a house keepinggene such as beta-actin or GAPDH or a plasmid carrying the specific“amplicon of interest”.

If polypeptide levels are to be monitored, any assay technique can beused that can determine levels of a specific polypeptide, includingradioimmunoassays (RIA), competitive-binding assays, Western Blotanalysis, FACS and enzyme-linked immunosorbent (ELISA) assays.Antibodies which specifically bind to particular splice isoforms may beused, for example. The antibodies may be used with or withoutmodification, and may be labelled by joining them, either covalently ornon-covalently, with an analytically-detectable reagent such as aradioisotope, a fluorescent molecule or an enzyme or other reportermolecule. A wide variety of reporter molecules known in the art may beused.

E1B Isoforms

Very little is known about the E1B-156R, E1B-93R, E1B-84R isoforms.Production of different E1B mRNAs is regulated during the infectionprocess. While mainly the 2.28 kb form is produced early in infection,the proportion of shorter spliced mRNAs increases over time and theE1B-84R transcript becomes predominant in the late phase of infection.The isoform protein expression closely follows the transcription patternof the mRNAs (Chow et. al (1979) J. Mol. Biol. 134:265-303; Montell et.al (1984) Mol. Cell. Biol. 4:966-972; Spector et. al (1978) J. Mol.Biol. 126:395-414; Virtanen and Pettersson (1985) J. Vivol. 54:383-391;Wilson and Darnell (1981) J. Mol. Biol. 148:231-251).

It has been shown that different spliceotypes can interact both hetero-and homogeneously with each other through the N-terminus, and theC-terminus must carry specific functions that cannot be complemented forby alternative E1B spliceotypes. When infecting with viruses lacking theexpression of a specific spliceotype the viability loss can becomplemented by co-transfecting with an expression plasmid forcorresponding spliceotype. The co-transfection with an alternativespliceotype does not complement the loss.

Mutant Adenoviruses

The table below summarises the details of some representative adenovirusmutants in provided as examples of the teaching of the invention, alongwith some experimental control viruses.

Virus Mutations^(a) Description Ixovex A3216G E1B-93R splice site mutantDoes not express E1B-93R isoform Destabilises E1B-496R due to sequencechange Increases in E1B-156R levels Ixo-ctrl Control (wild-type) virusIxo-156 T3272G/ E1B-156R splice acceptor site mutant G3275A Does notexpress E1B-156R isoform E1B-496R sequence is not changed Expressesother E1B gene products 93R levels increase Ixo-93 G3218A/ E1B-93Rsplice acceptor site mutant G3221A Does not express E1B-93R isoformE1B-496R sequence is not changed Increases in E1B-156R levels Ixo-SDG2255A/ E1B splice donor 1 site mutant T2258C Does not express E1B-93R,-156R and -84R isoforms E1B-496R sequence is not changed Ixo-Stop G2274TInserts a stop codon downstream of the E1B splice donor 1 site Does notexpress E1B-496R protein Expresses E1B-93R, -156R and -84R isoforms^(a)Numbered according to position in the Ad5 genome accession numberAC_000008.1 (SEQ ID NO: 41)

The adenovirus herein termed the Ixovex virus has diminished orinhibited expression of E1B-93R, and preferably does not expressE1B-93R. By “does not express” is meant herein that the detectable levelof the E1B-93R sequence is less than 50%, 10%, 1% of the level of theE1B-93R sequence in the wild type adenovirus under equivalentconditions, preferably less than 0.1%, less than 0.01% or even less.This has the effect of raising the expression of E1B-156R.

Additionally, in this virus the full length E1B protein E1B-496R isdestabilized. By “destabilised” is meant that the protein becomessubstantially undetectable due to the mutation. This leaves onlyE1B-156R and E1B-84R still expressed from the 496 reading frame. Theunstable nature of E1B-55k is discussed in Gabler et al. 1998 J. Virol.;and Gonzalez 2002, J. Virol.

The efficacy of Ixovex as compared to H101 suggests that to some extent,E1B-496R and E1B-156R have overlapping functions (Sieber et. al. (2007)J. Virol. 81 (1): 95-105). E1B-496R and E1B-156R have been found to bindmany similar factors. E1B-156R can bind to E4orf6, the binding partnerwith which E1B-496R utilises most of its important functions.Interestingly, E1B-156R has also been found to bind p53, although withless affinity. E1B-156R can substitute for E1B55k in cell transformationexperiments. Also, E1B-156R induces tumours in in vivo models, whenoverexpressed together with E1A. Specifically, the E1B-156R spliceomerwas found herein to have a cell transforming potential separate from theE1B-496R protein.

It is an advantage of the present invention that in order to achieve thedescribed oncogenic effect, viruses according to the present inventiondo not require the deletion of the whole of the E1B gene.

Methods of Generating Recombinant Viruses

The invention provides polynucleotides encoding the recombinantadenoviruses, optionally encoded within a vector suitable for virusproduction in a host cell.

The invention provides host cells comprising polynucleotides encodingthe recombinant adenovirus.

The invention also includes a method of rendering an adenovirusoncolytic. Such a method involves engineering a mutant adenovirus inwhich the sequence of the E1B gene has been modified so as to increasethe level of the E1B-156R isoform relative to the level in theequivalent wild-type adenovirus. The adenovirus type can be any of thosedescribed above, and is preferably a human adenovirus of subfamily groupC, namely one of serotypes 1, 2, 5, 6, or 57, even more preferably, theterm adenovirus applies to two human serotypes, Ad2 and Ad5. Similarly,the mutation may be any one of those described or exemplified herein. Incertain embodiments, a hybrid virus may be engineered, for example, inwhich an E1B-156R from one adenovirus variant is expressed in anotheradenovirus variant. For example, an Ad2 E1B-156R may be expressed in anAd5 adenovirus; it has been shown herein that adding Ad2 E1B-156R to Ad5increases oncolytic activity by 10-fold.

Suitable techniques to engineer mutations in alternative adenoviruseswill be known to those of skill in the art. A preferred method could beto use the widely used pShuttle system (Agilent Technologies) or use themethod developed by Dr. Oberg (the inventor of IXOvex and board memberof IXOgen)) using the pSuperShuttle system (see Ingemarsdotter, C. K.,S. K. Baird, C. M. Connell, D. Oberg, G. Hallden, and I. A. McNeish.2010. Low-dose paclitaxel synergizes with oncolytic adenoviruses viamitotic slippage and apoptosis in ovarian cancer. Oncogene29:6051-6063). This allows the insertion or mutation of any sequenceanywhere in the adenovirus, which pShuttle cannot do, being limited tothe end regions of the adenoviral genome. Shortly, the flankingsequences (left and right arm) of the region of interest may be clonedinto the pSuperShuttle plasmid on each side of an antibiotic selectiongene (ASG). If a mutation of any sort (substitution, deletion oraddition) is desired it can be incorporated in either arm. For theinsertion of a gene of interest or a whole expression cassette into thevirus the extensive multiple cloning sites on each side of the ASG maybe used. When the complete pSupershuttle construct is sequenced andready it is fused with the virus by homologous recombination. Theinserted ASG allows for positive selection. The ASG is digested awayleaving a small scar in the form of a unique restriction enzyme site,which can be used in future modifications of the virus. Other suitablevariations on this technique will be known to those of skill in the art.

Construction of Adenovirus E1B-55K Mutants

Methods for the construction of adenoviral mutants are generally knownin the art. See, Mittal (1993) Virus Res., 28: 67-90 and Hermiston etal., Methods in Molecular Medicine: Adenovirus Methods and Protocols(1999) ed. Wold, Humana Press. Further, the adenovirus 5 genome isregistered as NCBI Reference Sequence: AC_(—)000008.1, and the virus isavailable from the American Type Culture Collection, Rockville, Md.,U.S.A., under accession number: VR-1516.

Generally, adenovirus vector construction involves an initial deletionor modification of a desired region of the adenoviral genome, preferablythe Ad5 genome, in a plasmid cassette using standard techniques.

Certain of the materials and methods used to construct adenovirusmutants are described by Hanke et. al. (1990) Virology, 177: 437-444 andBett et. al., (1993) J. Virol. 67: 5911-5921, and in PCT/CA96/00375.Many of the materials used to construct adenovirus mutants are providedcommercially. See also, Hermiston et al., Methods in Molecular Medicine:Adenovirus Methods and Protocols (1999) ed. Wold, Humana Press. Otherdetails are provided herein.

Cell lines that were used to conduct the experiments described hereinare readily available from recognised depositary institutions. Forexample, the following cell lines were used herein to assesscytotoxicity: H1299, FaDu, H460, A549, HeLa, Hek293, JH293 and NHBE.

A preferred procedure for constructing the adenoviral E1B gene mutantsof the present invention is to make site-specific mutations in theadenoviral genome in a plasmid cassette using well establishedtechniques of molecular biology, or modifications of these techniques,referred to herein. This can be realized using various materials andmethods.

Methods of Treating Cancer

The invention provides recombinant adenoviruses that produce anoncolytic effect in a cancer cell. The cancer cell may be a neoplasticcell. The invention also provides novel methods of treating cancer,characterised by neoplastic cells. The neoplastic cells may preferablysubstantially lack p53 function (p53⁽⁻⁾). Such a method may comprise:

a) administering a dose of the recombinant adenovirus according to theinvention, that carries a mutation in the E1B gene, to a patient in needof treatment;

b) allowing sufficient time for the recombinant adenovirus to infectneoplastic cells of said cancer, wherein the mutant adenovirus has anoncolytic effect which is selective for the cancer cells relative to thenon-neoplastic cells; and

c) optionally administering further doses of the recombinant adenovirus.

The cancer cell or neoplastic cell may substantially lack p53 function.

The invention provides recombinant adenoviruses for use as a therapeuticagent in treating a patient with cancer. Preferably the cancer ischaracterised by neoplastic cells. Preferably, those neoplastic cellssubstantially lack p53 function.

The invention also provides compositions comprising recombinantadenoviruses of the invention.

The invention provides pharmaceutical compositions comprising arecombinant adenovirus of the invention.

The invention also provides processes for making a pharmaceuticalcomposition involving combining a recombinant adenovirus of theinvention with a pharmaceutically acceptable carrier.

The compositions may additionally comprise an agent for chemotherapy.

The present invention provides several novel methods and compositionsfor ablating neoplastic cells by infecting the neoplastic cells with arecombinant adenovirus which is substantially replication-deficient innon-neoplastic cells and which exhibits at least a partial replicationphenotype in neoplastic cells. The difference in replication phenotypeof the adenovirus constructs of the invention in neoplastic andnon-neoplastic cells provides a biological basis for viral-based therapyof cancer.

A cell population (such as a mixed cell culture, human cancer patient ornon-human mammalian subject) which comprises a subpopulation ofneoplastic cells lacking p53 function and a subpopulation ofnon-neoplastic cells which express essentially normal p53 function canbe contacted under infective conditions (i.e. conditions suitable foradenoviral infection of the cell population, typically physiologicalconditions) with a composition comprising an infectious dosage of aE1B-p53⁽⁻⁾ replication inhibited adenovirus. Such a contacting stepresults in infection of the cell population with the E1B-p53⁽⁻⁾replication-deficient adenovirus. The infection produces preferentialexpression of a replication phenotype in a significant fraction of thecells comprising the subpopulation of neoplastic cells lacking p53function, but does not produce a substantial expression of a replicativephenotype in the subpopulation of non-neoplastic cells havingessentially normal p53 function. The expression of the replicationphenotype in an infected p53⁽⁻⁾ cell results in the death of the cell,such as by the cytopathic effect (CPE), cell lysis, apoptosis, orsimilar, resulting in a selective ablation of neoplastic p53⁽⁻⁾ cellsfrom the cell population.

It is desirable for the mutant virus to be replicable and to forminfectious virions containing the mutant viral genome which may spreadand infect other cells, thus amplifying the anti-neoplastic action of aninitial dosage of mutant virus.

Herein, E1B-p53⁽⁻⁾ replication inhibited adenovirus constructs suitablefor selective killing of p53⁽⁻⁾ neoplastic cells are those describedabove.

Candidate antineoplastic adenovirus mutants may be further evaluated bytheir capacity to reduce tumourigenesis or neoplastic cell burden innu/nu mice harbouring a transplant of neoplastic cells lacking p53function, as compared to untreated mice harbouring an equivalenttransplant of the neoplastic cells.

Antineoplastic replication-deficient adenovirus mutants may beformulated for therapeutic, prophylactic and, potentially, diagnosticadministration to a patient having a neoplastic disease. For therapeuticor prophylactic uses, a sterile composition containing apharmacologically effective dosage of one or more species ofantineoplastic replication inhibited adenovirus mutant is administeredto a patient for treatment of a neoplastic condition. A pharmaceuticallyacceptable carrier or excipient is often employed in such sterilecompositions. A variety of aqueous solutions can be used, e.g., water,buffered water, 0.4% saline, 0.3% glycine and the like. These solutionsare sterile and generally free of particulate matter other than thedesired adenoviral virions. The compositions may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions such as pH adjusting and bufferingagents, toxicity adjusting agents and the like, for example sodiumacetate, sodium chloride, potassium chloride, calcium chloride, sodiumlactate, etc. Excipients which enhance infection of cells by adenovirusmay be included.

Therapy of neoplastic disease may be afforded by administering to apatient or subject a composition comprising replication-defectiveadenoviruses of the invention. Various human and mammalian neoplasmscomprising cells that lack p53 function may be treated with thereplication inhibited adenoviral constructs. For example (but notlimiting to), a human patient or non-human mammal having a bronchogeniccarcinoma, nasopharyngeal carcinoma, laryngeal carcinoma, small cell andnon-small cell lung carcinoma, lung adenocarcinoma, hepatocarcinoma,pancreatic carcinoma, bladder carcinoma, colon carcinoma, breastcarcinoma, cervical carcinoma, ovarian carcinoma, or lymphocyticleukaemias may be treated by administering an effective antineoplasticdosage of an appropriate replication inhibited adenovirus.

Suspensions of infectious adenovirus particles may be applied toneoplastic tissue by various routes, including intravenous,intraperitoneal, intramuscular, subdermal, and topical. A adenovirussuspension, preferably an aqueous suspension, containing between about10³ to 10¹⁵ or more virion particles per ml (such as between about 10⁵to 10¹² or more virion particles per ml, between about 10⁷ to 10¹⁰ ormore virion particles per ml, or about 10⁹ virion particles per ml) maybe inhaled as a mist (e.g. for pulmonary delivery to treat bronchogeniccarcinoma, small-cell lung carcinoma, non-small cell lung carcinoma,lung adenocarcinoma, or laryngeal cancer). Alternatively, such asuspension may be swabbed directly on a tumour site for treating atumour (e.g. bronchogenic carcinoma, nasopharyngeal carcinoma, laryngealcarcinoma, cervical carcinoma) or may be administered by infusion (e.g.into the peritoneal cavity for treating ovarian cancer, into the portalvein for treating hepatocarcinoma or liver metastases from othernon-hepatic primary tumours) or other suitable route, including directinjection into a tumour mass (e.g. a breast tumour), enema (e.g. coloncancer), or catheter (e.g. bladder cancer).

Replication inhibited viruses may also be delivered to neoplastic cellsby liposome or immunoliposome delivery; such delivery may be selectivelytargeted to neoplastic cells on the basis of a cell surface propertypresent on the neoplastic cell population (e.g. the presence of a cellsurface protein which binds an immunoglobulin in an immunoliposome). Forexample, a suspension of replication inhibited adenovirus virions can beencapsulated in micelles to form immunoliposomes by conventional methods(for example see U.S. Pat. No. 5,043,164, U.S. Pat. No. 4,957,735, U.S.Pat. No. 4,925,661; Connor and Huang (1985) J. Cell Biol. 101: 582;Lasic D D (1992) Nature 355: 279; Novel Drug Delivery (1989) eds.Prescott and Nimmo, Wiley, New York; and Reddy et al. (1992) J. Immunol.148: 1585). Immunoliposomes comprising an antibody that bindsspecifically to a cancer cell antigen (e.g., CALLA, CEA) present on thecancer cells of the individual may be used to target virions to thosecells.

The compositions containing the present antineoplasticreplication-deficient adenoviruses or cocktails thereof can beadministered for prophylactic and/or therapeutic treatments ofneoplastic disease. In therapeutic application, compositions areadministered to a patient already affected by the particular neoplasticdisease, in an amount sufficient to cure or at least partially arrestthe condition and its complications. An amount adequate to accomplishthis is defined as a “therapeutically effective dose” or “efficaciousdose.” Amounts effective for this use will depend upon the severity ofthe condition, the general state of the patient, and the route ofadministration.

In prophylactic applications, compositions containing the antineoplasticreplication inhibited adenoviruses or cocktails thereof are administeredto a patient not presently in a neoplastic disease state to enhance thepatient's resistance to recurrence of a neoplasm or to prolong remissiontime. Such an amount is defined to be a “prophylactically effectivedose.” In this use, the precise amounts again depend upon the patient'sstate of health and general level of immunity.

Single or multiple administrations of the compositions can be carriedout with dose levels and pattern being selected by the treatingphysician. In any event, the pharmaceutical formulations should providea quantity of the antineoplastic replication inhibited adenoviruses ofthis invention sufficient to effectively treat the patient.

Antineoplastic replication inhibited adenoviral therapy of the presentinvention may be combined with other antineoplastic protocols, such asconventional chemotherapy.

General

The term “comprising” encompasses “including” as well as “consisting”e.g. a composition “comprising” X may consist exclusively of X or mayinclude something additional e.g. X+Y.

The word “substantially” does not exclude “completely” e.g. acomposition which is “substantially free” from Y may be completely freefrom Y. Where necessary, the word “substantially” may be omitted fromthe definition of the invention.

The practice of the present invention will employ, unless otherwiseindicated, conventional techniques of molecular biology, microbiology,recombinant DNA technology and immunology, which are within the skill ofthose working in the art.

Most general molecular biology, microbiology recombinant DNA technologyand immunological techniques can be found in Sambrook et al., MolecularCloning, A Laboratory Manual (2001) Cold Harbor-Laboratory Press, ColdSpring Harbor, N.Y. or Ausubel et al., Current protocols in molecularbiology (1990) John Wiley and Sons, N.Y.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1. Schematic description of an oncolytic virus.

FIG. 2. E1B splice map showing alternative E1B gene products. Thefull-length E1B transcript carries the E1B-176R and E1B-496R openreading frame (ORF). Through alternative splicing in the E1B-496 ORFanother three proteins, E1B-93R, E1B-156R and E1B-84R, are expressed.The lesser proteins have the 79 amino terminal amino acids in commonwith E1B-496R but differ in the carboxy terminal, except for E1B-156R,which splices in frame with E1B-496R.

FIG. 3. An amino acid change in Ixovex in the E1B-55k protein inhibitsits expression. A549 cells were infected with the respective virus at 5pfu/cell and total cell lysate was collected 48 hours post infection(hpi). Shown is a western blot stained with polyclonal α-capsid proteinAb6982 antibody (top panel), monoclonal α-E1B-55k antibody 2A6 (middlepanel) and monoclonal α-actin antibody I-19-SC as loading control(lowest panel).

FIG. 4. The point mutation in the E1B55k open reading frame in Ixovexinhibits splicing to the 93R splice acceptor. A549 cells were infectedwith the respective virus at 5 pfu/cell and total RNA was collected at48 hpi. cDNA was made using an oligo-dT primer. PCR was performed usinga common sense primer upstream of the 55k splice donor and specificprimers downstream of respective splice acceptor. The PCR reactions wererun on a 2% agarose TBE gel stained with GelRed.

FIG. 5. Ixovex is inhibited in inducing degradation of p53. A549 cellswere infected with the respective virus at 5 pfu/cell and cell lysatewas collected 48 hpi. Shown is a western blot staining with a monoclonalα-p53 antibody #9282 CS (top panel) and the monoclonal α-actin antibodyI-19-SC as loading control (lower panel).

FIG. 6. Replication assay in cancer cell lines. Each cell line wasinfected with 5 pfu/cell of respective virus. Cells and media wereharvested at 24, 48 and 72 hpi and analysed by a limited dilution assay.CPE was noted visually after 10 days and TCID₅₀ (pfu/cell) results werecalculated, as described in materials and methods.

FIG. 7. The relative cytotoxicity of the Ixovex, Ad5wt and Onyx-015viruses in cancer cells. The respective cell line was infected with theindicated viruses in a 5-fold dilution series. The cytotoxicity wasmeasured 6 days post infection (dpi) using the MTS assay and EC₅₀ valueswere calculated.

FIG. 8. Replication efficiency in Normal Human Bronchial Epithelialcells. Each cell line was infected with 5 pfu/cell of the respectivevirus. Cells and media were harvested at 24, 48 and 72 hpi and analysedby a limited dilution assay. CPE was noted visually after 10 days andTCID₅₀ (pfu/cell) results were calculated, as described herein.

FIG. 9. Ixovex shows more than 500-fold less cytotoxicity to normalcells compared with the unmodified virus (Adswt). Presented is the foldinhibition of cytotoxicity in relation to Ad5wt (bottom row) and the rawEC₅₀ values (top row). The cytotoxicity was measured at 6 dpi using theMTS assay.

FIG. 10. The E1B-156R protein is overexpressed by Ixovex. Western blotwas performed on total protein extracted at 48 hpi from H1299 cellsinfected with 10 pfu/cell of Ixovex or Ad5wt.

FIG. 11. Ad5- and Ad2-156R proteins enhanced the Oncolytic Index (OI) incancer cells as compared to normal cells. A) Ad5- and Ad2-156Rexpression plasmids were transfected into Ad5wt-infected HeLa and NHBEcells. In parallel, an additional cancer cells line (H460, large celllung carcinoma) was included in Ad5-156R complementation. B) Ad5-156Rwas transfected into ONYX-015 infected cells. C) Ad5-156R wastransfected into Ad2wt-infected cells. The cells were infected with 2.5pfu/cell and complemented or cross-complemented with expression plasmidsfor the respective E1B-156R. Samples were analysed with a Burst (viralreplication) assay, at the indicated hpi.

FIG. 12. Table showing the 48 hpi data points for FIGS. 11A-C (exceptfor the H460 data points which were collect 72 hpi) and calculation ofoncolytic indices, where OI=((a/b)/(c/d)). a=pfu/cellcancer cells+156R,b=pfu/cellnormal cells+156R, c=pfu/cellcancer cells−156R,d=pfu/cellnormal cells−156R.

FIG. 13. The protein sequence of E1B-156R for the serotypes ofadenovirus subfamily C aligned with the sequences for serotypes of thesubfamilies B, D and E. Shaded field: The similarities within group C.Gaps indicates where they differ. Underlined on Ad5: The amino acidsthat would single out Ad5-156R from the other E1B-156R proteins insubfamily C.

FIG. 14. DNA gels showing amplified cDNA bands corresponding to E1B-156Rin Ad2, Ad4 and Ad11. All bands in the gels were cloned into the Topo-IIPCR Blunt Vector (Invitrogen) and sequenced to confirm the indicatedbands corresponded to the E1B-156R of each respective virus.

EXAMPLES

Materials and Methods

Virus Construction

Nucleotides 1-5055 of adenovirus serotype 5 (Ad5) were PCR amplifiedwith Phusion PFU polymerase using Ad5start (SEQ IDNO:15—ccacctcgagttaattaacatcatcaataatataccttattttg) and Ad5wt5055as (SEQID NO: 16—gtgggtttaaacggatttggtcagggaaaacatg) oligonucleotides. Viralgenomic DNA extracted from a CsCl purified Ad5 batch was used as atemplate. The PCR product was cloned into pShuttle (Stratagene) usingrestriction enzymes NotI and PmeI (NEB). To produce E1B 93R splice sitemutations in pShuttle-5055, the oligonucleotides Mut93Rs (SEQ ID NO:17—ccttgcatttgggtaatagaagaggagtgttcctaccttaccaatg) and Mut93Ras (SEQ IDNO: 18—cattggtaaggtaggaacactcctcttctattacccaaatgcaagg) were used in aPCR Mutagenesis XL reaction (Stratagene), according to manufacturers'instructions. Clones were screened and sent for sequencing. Five μg ofthe correct clone were linearised using PmeI (NEB),phenol/chloroform-treated and ethanol precipitated. Two hundred ng weremixed together with 100 ng of the pTG3602 plasmid. The mixture waselectroporated into BJ5183 cells (Stratagene) and plated onto kanamycin(25 μg/ml) containing agar-plates. Clones were screened by sizeexclusion on a cracking gel. Briefly, the pellet of 1 ml bacterialculture was resuspended in 50 μl water and treated with 50 μlphenol/chloroform. The mixture was spun for 1 min at 13,000 rpm and thewater phase collected. The water phase containing all DNA and RNA fromthe bacteria was treated for 5 min with DNA loading dye containingRNaseH and then run on a 0.7% agarose gel. DNA was prepared from theselected clones (Qiagen Maxi Prep kit) and sequenced to ensure thecorrect mutation had been introduced. Five μg of correct pT3602 mutantwere digested with PacI to excise the viral genome, phenol/chloroformtreated and ethanol precipitated. Two μg of the digested plasmid weretransfected into 10e5 Hek293 cells in a 6-well plate using Transfectene(Biorad). Five days later the cells were harvested, subjected to threerounds of freeze/thawing and applied to a T175 bottle 80% subconfluentwith A549 cells for bulking up of infected cell lysate.

A CF-10 (Thermo Scientific) was seeded with Hek293 cells and infected at80% confluency with a third of the cell lysate. Three days later theCF-10 was harvested. The cell pellet was freeze/thawed three times,centrifuged to clear the lysate and applied to a 1.25/1.4 g/ml CsClgradient and spun at 25,000 rpm in an ultracentrifuge. The virus bandwas collected with a 21 G syringe and distributed into 1.35 g/ml CsClcolumns. The columns were spun at 40,000 rpm overnight and the virusband was collected with a 21 G syringe. The extracted virus was injectedinto a Slide-A-Lyzer (Thermo Scientific) cassette and dialysed overnightat 4° C. into 50 mM TRIS pH 7.8, 150 mM NaCl, 1 mM MgCl₂, 10% glycerol.The virus activity, assessed by the 50% tissue culture infective dose(TCID₅₀) (pfu/ml), was then determined by using JH293 cells as describedin the Viral Replication section below. Viral DNA was purified from asmall aliquot and the number of viral genomes per μl (particles/μl) wasdetermined using a spectrophotometer. The ratios between the particlesand activities of all viruses used herein were less than 20. Ixo-ctrlvirus is a wild-type clone from the adenoviral serotype 5 strainpTG3602.

In parallel, pShuttle plasmids were made in which all splice sites wereindividually mutated without changing the amino acid sequence of theE1B-496R protein, using the PCR Mutagenesis XL Kit (Stratagene)according to manufacturers' recommendations.

Oligonucleotides   for PCR Oligonucleotide SEQ ID Mutagenesis sequencesNO: Ixo-CtrlS (wt) CTTGCATTTGGGTAACAGg SEQ ID aggggggtgttcctacc NO: 19Ixo-CtrlAS (wt) ggtaggaacacccccctc SEQ ID CTGTTACCCAAATGCAAG NO: 20Ixo-156Rs ctaaGATATTGCTgGA SEQ ID acccgagagcatgtcc NO: 21 Ixo-156RasggacatgctctcgggtTCc SEQ ID AGCAATATCttag NO: 22 Ixo-93Rs CATTTGGGTAACAGSEQ ID aagaggggtgttcc NO: 23 Ixo-93Ras ggaacacccctctt SEQ IDCTGTTACCCAAATG NO: 24 Ixo-SDs gaatgaatgttgtacaaGTc SEQ ID GCTGAACTGTATCNO: 25 Ixo-SDas GATACAGTTCAGCgACttgt SEQ ID acaacattcattc NO: 26Ixo-StopS gtggctgaactgtatccataac SEQ ID tgagacgcattttg NO: 27 Ixo-StopAScaaaatgcgtctcagttatgg SEQ ID atacagttcagccac NO: 28 IxovexSenseCATTTGGGTAACGGG SEQ ID aggggggtgttcc NO: 39 IxovexAS ggaacacccccctSEQ ID CCCGTTACCCAAATG NO: 40

All these pShuttle plasmid were used in homologous recombination togenerate a large set of viruses (see table 1).

TABLE 1 Adenovirus mutants provided by the invention. VirusMutations^(a) Description Ixovex A3216G E1B-93R splice site mutant Doesnot express E1B-93R isoform Destabilises E1B-496R due to sequence changeIncreases in E1B-156R levels Ixo-ctrl Control (wild-type) virus Ixo-156T3272G/ E1B-156R splice acceptor site mutant G3275A Does not expressE1B-156R isoform E1B-496R sequence is not changed Expresses other E1Bgene products 93R levels increase Ixo-93 G3218A/ E1B-93R splice acceptorsite mutant G3221A Does not express E1B-93R isoform E1B-496R sequence isnot changed Increases in E1B-156R levels Ixo-SD G2255A/ E1B splice donor1 site mutant T2258C Does not express E1B-93R, -156R and -84R isoformsE1B-496R sequence is not changed Ixo-Stop G2274T Inserts a stop codondownstream of the E1B splice donor 1 site Does not express E1B-496Rprotein Expresses E1B-93R, -156R and -84R isoforms ^(a)Numberedaccording to position in the Ad5 genome accession number AC_000008.1(SEQ ID NO: 41).

Tissue Culture

All cells were cultured at 37 C and 5% CO₂ and were tested regularly formycoplasma contamination. The cell lines used in this study are listedbelow.

Cell name Type Culture medium Source H1299 Non-small cell lung DMEM +10% FCS BCI* carcinoma FaDu Pharyngeal squamous cell DMEM + 10% FCS BCIcarcinoma H460 Large cell lung carcinoma DMEM + 10% FCS BCI A549Non-small cell lung DMEM + 10% FCS Uppsala carcinoma University HeLaCervical Cancer DMEM + 10% FCS BCI Hek293 Human Embryonal Kidney DMEM +10% FCS Uppsala cells University JH293 Human Embryonal Kidney DMEM + 10%FCS Uppsala cells University NHBE Normal Human Bronchial Bullet Kit(Lonza) Lonza Epithelial

Cytotoxicity Assay

We used the 3 -(4,5 -dimethylthiazol-2-yl)-5 (3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolim (MTS) assay(CellTiter 96 Aqueous Non-Radioactive Cell Proliferation Assay; Promega,Wis., USA) to assess the cytotoxicity of Ixovex and the control viruses.Aiming for cells to be confluent on day 6, 1,000-4,000 cells/well(depending on the rate of growth) were seeded in a 96 well plate in 90μl of medium and 5% FCS. Viruses (in 10 μl of medium and 5% FCS) wereadded 18 hours later at nine 1:10 serial dilutions starting at 10,000viral particles (vp)/cell, together with a positive (just cells with novirus) and a negative control (no cells just medium).

Six days following infection, survival was determined using MTS assay.MTS was mixed with phenazinemethosulphate (PMS) at 20:1 ratio and addedto the cells. Following three hours of incubation, absorbance wasmeasured at 490 nm using the Opsys MR 96-well μplate reader andRevelation Quicklink 4.04 software (Dynex Technologies, Virginia, USA).The values were established for each dilution and compared to negativecontrol (100% cell death), and positive control (0% cell death). EC₅₀values (half maximum effective concentration to kill 50% of cells—EC₅₀)were calculated by non-linear regression (sigmoidal dose-response curve)using GraphPad Prism (GraphPad Software, California, USA), utilising thefollowing formula:

Y=bottom+(top-bottom)/1+10^(┌log) ¹⁰ ^(EC) ⁵⁰ ^(−X)×Hill slope┐)

Y is the response and starts at ‘bottom’ and goes to the ‘top’ in asigmoidal fashion.

All experiments were performed in triplicate.

Viral Replication Assay

Cells were seeded in 6-well plate in medium with 10% FCS 24 hours priorto infection. 100 vp/cell were used to infect 80% confluent cells in a2% FCS medium. Two hours after infection, the medium was replaced with a10% FCS medium (primary infection). At hours post-infection (specifiedin respective figure), medium and cells were harvested (by scraping),frozen and thawed three times in liquid nitrogen and 37° C.,respectively and stored at −80° C. until used. JH293 cells were seededat 10,000 cells per well in a 96-well plate in 200 μl medium with 5%FCS. In the first row of the TCID50 the initial dilution of thedifferent samples was between undiluted to 1:1000 dependent on hpi andvirus, these dilutions from the primary infection were used to infectJH293 cells. The last row was left uninfected as a negative control.Between day 9 and 11, plates were inspected for cytopathogenic effect(CPE). The 50% tissue culture infective dose (TCID₅₀) and number ofpfu/cell (cell count on the day of infection) were calculated usingReed-Muench accumulative method. See example below:

Example of a 96-well plate(+indicate well with evidence of CPE):

% with Dilution CPE 10⁻³ + + + + + + + + + + + + 100%10⁻⁴ + + + + + + + + + + + + 100% 10⁻⁵ + + + + + + + + + + + + 100%10⁻⁶ + + + + 42% 10⁻⁷ 0% 10⁻⁸ 0% 10⁻⁹ 0% Nega- tive controls Calculatethe proportionate distance: (% next above 50% − 50%)/(% next above 50% −% next below 50%) = (100% − 50%)/(100% − 42%) = 0.86 Calculate the 50%end point: log₁₀ (dilution in which position is next above 50%) = log₁₀10⁻⁵ = −5 Combine the values to obtain log₁₀ TCID₅₀ = −5 − 0.86 = −5.86TCID₅₀ titre = 10^(−5.86) (or 1 in 7.24 × 10⁵ dilution of the amountadded to the top row). As 22 μl (0.022 ml) was added to the top row,TCID₅₀/ml = 7.24 × 10⁵/0.022 = 3.29 × 10⁷ Multiple by a constant: 3.29 ×10⁷ × 0.69 = 2.27 × 10⁷ pfu/ml

For pfu/cell, multiply the above with the volume of virus added to eachwell of the 6-well plate (2 ml) and divide by the cell count on the dayof infection (e.g. 2.4×10⁵): (2.27×10⁷×2)/2.4×10⁵=189 pfu/cell.

Western Blot

A549 cells or H1299 were infected with 5 pfu/cell, total protein wasextracted at 48 h post infection using RIPA buffer. Proteinconcentration was determined using Bradford reagent. Twenty μg totalprotein from each sample were loaded onto a 10% PAGE gel. The proteinswere transferred to a PVDF (BioRad) membrane by wet blotting. Themembrane was blocked using 3% BSA TBS solution for 1 h. Primaryantibodies used were: Ad capsid proteins (AbCam-6982), E1B-55k (2A6,Sarnow, Sullivan Levine 1982, dilution 1:500), E1A (Santa cruz, M73),actin (Santa Cruz, 1-19) and p53 (Cell Signalling, #9282). Allantibodies were diluted as recommended in 1.5% BSA TBS. Membranes wereincubated with the primary antibodies for 15-24 hours at 4° C. whereafter they were washed with 1×TBS 3% Tween-20 three times for 10 min.HRP-coupled secondary antibodies against respective primary antibodywere diluted 1:5000 in 1.5% BSA TBS and applied to the membrane for 1 h.After removing the antibody dilution the membranes were washed with1×TBS 3% Tween-20 three times for 10 min. Each membrane was exposed for1 min with ECL Plus (GE, RPN2132). After having been wrapped in plasticfoil the membranes were put in a Hypercassette together with Hyperfilm(GE) and the films were developed at selected time intervals.Alternatively, secondary antibodies labelled with IRDyes from LI-CORwere used. Analysis was carried out using an Odyssey Imager.

RT-PCR

A549 cells were infected with 5 pfu/cell of respective virus, total RNAwas extracted at 48 h post infection using Trizol (Invitrogen). The RNAwas DNAse treated (NEB, DNase I), phenol/chloroform treated and ethanolprecipitated. One μg total RNA was used to synthesise cDNA (Invitrogen,SuperScript® III) according to manufacturers' recommendations. cDNA wasused as template in PCR (NEB Taq DNA Polymerase) reactions with a commonsense oligonucleotide (55kSense, SEQ ID NO: 29—gcctgctactgttgtcttccg)and either of the following antisense nucleotides: 93Ras, SEQ ID NO:30—cacccccctcctgtacaac, 156Ras, SEQ ID NO: 31—gacatgctctcgggctgtacaac or84Ras, SEQ ID NO: 32 caaacgagttggtgctcatg. The amplicon length of eachwas about 200 nucleotides. The PCR reaction was stopped after 20 cyclesand an aliquot run on a 2% agarose gel.

Making the Adenoviral E1B-93R Splice Site Acceptor Mutant

The first 5000 nucleotides of the Ad5wt genome (NCBI Reference Sequence:AC_(—)000008.1) were PCR amplified (primers Ad5wt5000start: SEQ ID NO:15—ccacctcgagttaattaaCATCATCAATAATATACCTTATTTTG; Ad5wt5000as: SEQ ID NO:16—gtgggtttaaacGGATTTGGTCAGGGAAAACATG) and agarose gel purified. Thepurified product was digested with restriction enzymes NotI and PmeI(New England Biolabs) and cloned into the pShuttle plasmid (Stratagene),replacing the existing left arm for homologous recombination, producingpShuttle-LA, as by Agilent Technologies, AdEasyhttp://www.genomics.agilent.com/CollectionSubpageaspx?PageType=Product&SubPageType=ProductData&PageID=592. This plasmidwas recombined with the plasmid pTG3602, containing the complete Ad5wtgenome, as recommended by Agilent Technologies in their AdEasy systemusing BJ5183 recombination competent cells. After recombination theBJ5183 bacteria were plated onto agar plates containing kanamycin.Single colonies were picked, grown and DNA was prepared from largecultures. Each DNA preparation was screened for the correctrecombination event. The digestion of the genomes was performed with Pad(New England Biolabs). After the correct clones had been grown on agarplates, the genomes were digested out and transfected into HEK293 cellsusing Transfectin Transfection Reagent (Bio-Rad) according to themanufacturers' instructions. Four days after transfection virus lysateswere harvested [Cells were collected by scraping together with the mediaand collected in a 15 ml falcon tube. The sample was freeze/thawed threetimes and used to infect a T175 bottle about 90% confluent with Hek293cells. Three days later the cells and media was harvested andfreeze/thawed three times] and then used to infect viral productionfactories called CF-10s (Nunc). These have the approximate surface areaof forty T175 bottles, i.e. 7000 cm², and are used to grow large numberof cells for the production of a large number of viruses. Briefly, thecells of four confluent T175 bottles were transferred in 1 L of 5% FCSDMEM media into a CF10. Twenty-five ml (1/40) of the cell-containingmedia was applied to a new T175 as a growth control. On the day the T175was 90% confluent the CF10 was at the same stage. Half of the celllysate was then injected into the CF10 and the media was moved aroundfor an even distribution. Three days later the CF10 was shaken todislodge the cells that had not started floating around yet due to viralinfection. The CF10 was emptied, the cells spun down, washed in PBS andfinally suspended in 50 mM Tris-HCl, pH7.8. Purification was carried outby Caesium-chloride banding. Viruses were then purified and analysed fortitre (particles) and activity (pfu). The vp:pfu unit ratio (vp:pfuunits) for all viruses was between 10-20. Sequencing of one of the viralclones showed that it had a point mutation in the E1B-93R spliceacceptor site.

Confirming the Existence of E1B-156R in Ad2, Ad4 and Ad11

A549 cells were infected with 5 pfu/cell of each virus. At 48 hpi, totalRNA was extracted and 1 μg was converted to cDNA using Superscript-II(Invitrogen) with random hexamers. One μl of the total 50 ml was used astemplate in a PCR with serotype-specific primers (Ad2sense, SEQ ID NO:33—ctcgaggaattcgccaccatggagcgaagaaacccatc, Ad2antisense, SEQ ID NO.34—cacttctagatcaatctgtatcttcatcgctag, Ad4sense, SEQ ID NO.35—ggagataggacggtcttgg, Ad4antisense, SEQ ID NO.36—ggatcccatcacailligacg, Ad11sense, SEQ ID NO. 37—catccatggaggtttgggc,Ad11antisense, SEQ ID NO. 38—ccttaaaagaagcgtttccac). FIG. 14 shows a DNAgel showing the cDNA bands and highlights the bands corresponding to theE1B-156R cDNA. All bands on the gels were purified (NucleoSpin Gel andPCR Clean-up, Macherey-Nagel) and cloned into a Topo-II PCR Blunt Vector(Invitrogen). Clones were sent for sequencing. Ad2-156R and Ad5-156Rwere cloned into the p3×Flag-CMV-14 vector using EcoRI and XbaI.

Testing if the E1B-156R Protein Enhances Oncolytic Index

The cDNA for E1B-156R from Ad2 and Ad5 were PCR amplified using startand stop primers specific for each respective E1B-55k as discussedabove. The primers included an EcoRI in the start primer and a XbaI sitein the stop primer. The PCR fragments were digested with the two enzymesand ligated into p3×Flag-CMV-14 (Sigma-Aldrich). The constructs weresequenced for the correct insert.

Cancer cells (Hela and H460) and normal cells (NHBE) were transfectedwith 2 μg Ad2-156R or Ad5-156R expression plasmids (or a controlplasmid) and co-infected with Ad5wt, ONYX-015 or Ad2wt. The transfectionwas performed using JetPRIME reagent (POLYPlus) according tomanufacturers' instructions. Infection with the viruses was performed asdiscussed above. Briefly, each well in a 6-well plate was transfectedwith 2 μl JetPRIME reagent and 200 μl transfection buffer. Control wellswere transfected with 2 μg inert plasmid, in the form of pUC19.

Viral replication was measured at various time points post-infectionusing the assays described above. The data are shown in FIGS. 11A, B andC. Oncolytic index was calculated as shown in FIG. 12.

Results

E1B-55k Protein is Lost

Total protein lysates from Ad5wt, Onyx-015, Ixovex and Ixo-ctrl infectedA549 cells showed that at 48 hpi all viruses expressed late protein(FIG. 3, top panel), i.e. had reached the late phase of adenoviralreplication. Both Ixovex and Onyx-015 viruses expressed less lateproteins than the Ad5wt and Ixo-ctrl, mirroring the reduced replicationefficiency seen in FIG. 6 for A549 cells. The Ixovex single nucleotidepoint mutation (SNP, genomic location 3216), which changes the aminoacid at position 400 in the E1B-55k protein from an arginine to aglycine, induced its destabilisation (FIG. 3, middle panel). The reducedamount of E1B-55k in Ixo-ctrl compared to Ad5wt mirrors the slightlylower replication efficiency of Ixo-ctrl in the A549 cells (FIG. 6, A549panel).

Dynamics in the Usage of the E1B Splice Acceptors

The SNP also inhibited use of the E1B-93R splice acceptor (FIG. 4,second panel) by changing the putative splice acceptor sequence fromCAG:GA to CGG:GA. Interestingly, as a secondary effect to the inhibitionof the 93R splice site, there is a compensatory switch to use of theE1B-156R splice acceptor (FIG. 4, middle panel). In the absence of theE1B-93R splice acceptor mutation, i.e. Ixo-ctrl, the relative use of the93R splice site is restored (FIG. 4, lowest panel) as compared to theAd5wt virus (FIG. 4, top panel). Onyx-015 could not be used in thisexperiment since this virus is deleted in the whole of the E1B-55k generegion.

Ixovex is Unable to Induce Degradation of p53

Western blot analysis showed that the SNP in Ixovex inhibited the virusfrom inducing the degradation of p53 (FIG. 5). In the absence of theE1B-93R splice acceptor mutation (i.e. Ixo-ctrl), the virus's capacityto inhibit p53 was restored. Interestingly, there was a much higherexpression of p53 in the Onyx-015 infected cells.

Replication Efficiency in Cancer Cells

A replication assay was performed using Ad5wt, Onyx-015, Ixovex andIxo-ctrl viruses in A549, HeLa, H460, H1299 and FaDu cells. Thereplication efficiency of all the viruses was below the detection limitin FaDu cells. In A549 and HeLa cells all viruses showed replicationefficiency up to two orders of magnitude lower than the Ad5wt virus(FIG. 6, top panels). The replication attenuation was not seen, or wasmuch less pronounced, in H460 and H1299 cells (FIG. 6, lower panels). Inthe two more permissive cancer cell lines, 100- to 1000-fold more Ixovexvirus was produced, compared to Onyx-015.

Cytotoxicity in Cancer Cells

FIG. 7 shows the cytotoxicity of the Onyx-015 and Ixovex virusesrelative to the Ad5wt virus in cancer cells. In comparison to Onyx-015,Ixovex was more efficient in all the cells tested (apart from H460cells, in which Onyx-015 was 2.5-fold more toxic). The cytotoxicity ofthe Ixovex virus was similar to (or much higher than) the Ad5wt virus inthe A549, HeLa and H1299 cancer cells lines.

Toxicity Profile in Normal Cells

The cytotoxicity of the Ad5wt, Onyx-015, Ixovex and Ixo-ctrl viruses wasmeasured in NHBE cells (Normal Human Bronchial Epithelial cells). Asseen in FIG. 9, the Ad5wt, Onyx-015, and Ixo-ctrl viruses are relativelytoxic in normal cells as compared to cancer cells. The Ixovex virushowever, showed an EC₅₀ value of 23 pfu/cell, which was higher than itsEC₅₀ in the A549, HeLa and H1299 cancer cell lines. Ad5wt and Ixo-ctrlvirus had EC₅₀ values of 0.042 and 0.031 pfu/cell, respectively, whileOnyx-015 had 0.63 pfu/cell. Thus, Ad5wt was greater than 500-fold,Ixo-ctrl greater than 700-fold and Onyx-015 was greater than 35-foldmore toxic to normal cells than Ixovex.

Replication Efficiency in NHBE Cells

At 48 hpi, virus activity was 30-fold higher in Ixo-ctrl and Onyx-015and 500-fold higher in Ad5wt compared to Ixovex virus (FIG. 8). Thesedifferences were even more pronounced at 72 hpi, where virus activitywas 50-fold higher in Ixo-ctrl and Onyx-015 and over 2000-fold higher inAd5wt. In fact, these differences might even be more pronounced sinceIxovex replication barely reached detection limit at all time points.

Ixovex Overexpressed the E1B-156R Protein

The protein levels of E1B-156R, adenovirus capsid proteins and E1Aexpressed by Ad5wt- and Ixovex-infected H1299 cells were analysed bywestern blot. FIG. 10 shows that Ixovex expressed similar amounts of allviral proteins except for the E1B-156R protein, the levels of which wereincreased by more than 20-fold, as compared to Ad5wt.

E1B-156R Protein Enhances Oncolytic Index

We hypothesised that adding the E1B-156R protein in trans would enhancethe oncolytic index (OI) for Ad5wt if the E1B-156R protein wasresponsible for the oncolytic effect. Transfecting an Ad5-156Rexpression plasmid and co-infecting with Ad5wt increased the OI by4-fold using Hela and NHBE cells (FIG. 11A and FIG. 12). An increase inoncolytic index was also observed when the same experiment was performedin an alternative cancer cell line, H460 (large cell lung carcinoma).Addition of Ad5-156R to Ad5wt—infected cells also had an enhancingeffect on viral replication (FIG. 11A). Ad5-156R was transfected intocells co-infected with the ONYX-015 virus, which lacks the E1B-156R genecompletely. The addition of Ad-156R increased the OI of the ONYX-015more then 5-fold at the 48 hpi time point (FIG. 11B and FIG. 12).Similarly, addition of Ad5-156R to Ad2-infected cells increased OI15-fold. Adenovirus serotypes from the same subfamily have a very smalldifference in protein sequence in comparison (FIG. 13). The closestadenovirus family member to Ad5 is Ad2. Addition of Ad2-156R toAd5wt-infected cells increased OI almost 3-fold.

Discussion

It was early discovered that the RNA expressed from the adenovirus E1Bgene region had a complex splicing pattern. The full-length 2.28 kb longRNA is polycistronic carrying two overlapping reading frames. Thealternative usage of either an early weak or a strong down-streamtranslation start site produces E1B-19k and E1B-55k, respectively(Perricaudet, Akusjarvi et al. 1979; Bos, Polder et al. 1981). A commonsplice donor early in the 55k ORF is used to splice to three alternativesplice acceptors, 93R SA, 156R SA and 84R SA (Anderson, Schmitt et al.1984; Virtanen and Peltersson 1985; Anderson, Maude et al. 1987). The93R AS splices out of frame with the 55k ORF adding a 15 amino acidC-terminus. The 156R AS splices in frame with E1B-55k removing the 340middle amino acids. The 84R SA is a down-stream site adding 6 aminoacids to the common N-terminus.

When making a large set of gene-modified viruses based on the wild typeadenovirus serotype 5 strain pTG3602 one viral clone was mutated at asingle nucleotide position (SNP) in the E1B-55k gene region. Themutation was made inside the splice acceptor sequence of E1B-93R, ormore precisely, it changed the putative site from cag/ga to cGg/ga. Notonly did the mutation change the splice site but it also changed E1B-55kamino acid 400 from an arginine to a glycine. This virus has been namedIxovex. We have characterised this virus when it comes to oncolyticpotential, meaning, retaining replication capacity and cytotoxicity incancer cells while being inhibited on both accounts in normal cells.

Our results show that the mutation leads to a lack of expressed E1B-55kprotein in infected cells (FIG. 3). We believe this is because the aminoacid change destabilises the E1B-55k protein. Others have introducedamino acid changes into E1B-55k and several of these made the proteinlevel unstable. In addition, our mutation changes an importantnucleotide in the E1B-55k splice acceptor site 93R, which negatessplicing to that particular splice site (FIG. 4). To compensate, thesplicing appears to be re-directed to the E1B-156R splice acceptor. Withthe lack of E1B-55k in the infection, Ixovex's ability to inhibit theexpression of p53 is severely reduced (FIG. 5). The reduced level ofinduced p53 by Ixovex compared to Onyx-015 could have been because ofthe slightly lower replication efficiency of Ixovex in A549 cells, i.e.the cells are less affected, hence less p53 is expressed. Alternatively,and what we believe, the increased splicing to the 156R splice acceptor(FIG. 4) may also increase expression of the E1B-156R protein. The 156Rsplicing splices in-frame with the C-(carboxy)-terminal part of E1B-55k.This removes the middle 340 amino acids leaving the C-terminal 78 aminoacids fused to the N-(amino)-terminal 79 amino acids. The Dobner lab hasshown (Sieber and Dobner 2007) that the E1B-156R protein retains someability to inhibit p53 through its C-terminus. It is also possible thatE1B-156R retains other functions of the E1B-55k protein.

The E1B-55k and E1B-156R protein interacts with many similar factors(Sieber and Dobner 2007; Schreiner, Wimmer et al. 2010; Schreiner,Wimmer et al. 2011; Wimmer, Blanchette et al. 2012). E1B-55k has beenassigned several functions besides mediating the degradation of p53. Itis also connected to regulating the selective nuclear export of lateviral RNA (Dobner and Kzhyshkowska 2001; Flint and Gonzalez 2003) andinhibiting translation of cellular RNA while promoting viral RNAtranslation (Blackford and Grand 2009). The main functions of E1B-55kare mediated when the protein is in complex with another viral protein,the E4orf6. Interestingly, the E1B-156R protein has been shown tointeract with the E4orf6 protein (Sieber and Dobner 2007). The E1B-156Rmight compensate for some of these functions, which fit with theincreased expression of E1B-156R by Ixovex.

In normal cells, the toxicity of each virus largely mirrored respectivereplication capacity. The lack of toxicity and the almost completeshutdown of replication in normal cells indicate an astounding safetyprofile of Ixovex. That the Onyx-015 virus replicated better in normalcells than Ixovex is intriguing considering that the deletion theOnyx-015 virus carries removes all possibilities to express E1B-55k,-93R and -156R protein (Barker and Berk 1987). This indicates that it isthe imbalance of expression in the E1B region that had the extensiveimpact on the attenuation of Ixovex in normal cells in comparison to theother viruses. Interestingly, the difference in replication in normalcells between Onyx-015 and Ixo-ctrl on one hand and Ixovex on the otherwas not seen in the cancer cells. This indicates that the Ixovexinfection in normal cells has become non-permissive, i.e. there isprobably a major blockage early in infection giving the cells time toclear the virus, whereas the transformed state of cancer cellscompensates for the lack of some E1B-55k function(s).

The effect of the imbalanced E1B expression in cancer cells wasdifferent depending on cancer cell line. The cytotoxicity of Ixovex inthe two highly replication-permissive cell lines H1299 and H460 was lowwhile the cytotoxicity was high in the replication-attenuated celllines, A549 and HeLa. The reason for this is probably because of thetoxicity, the cells died before producing high numbers of virus.

The adenovirus family is divided into 7 genera, named A-G, with a totalof more than 65 different serotypes. Serotype 5 (Ad5) belongs to generaC. We believe that the splicing pattern seen in Ad5 is conserved amongall adenovirus serotypes and that the imbalance through splice sitemutation causing a very advantageous oncoselectivity for Ad5 would bemirrored in most if not all of the other serotypes. Our preliminaryexperiments show similar splicing patterns in representative virusesfrom each of the different genera (A-Ad12, B1-Ad3, B2-Ad11, C-Ad5,D-Ad37, E-Ad4 and F-Ad40).

The overall higher efficacy of the Ad5wt virus to all the other virusesis probably due to the wild type strain pTG3602 (Oberg, Yanover et al.2010), used as genome backbone for Ixovex and Ixo-ctrl. This backbonecarries a few point mutations scattered throughout the genome. OurIxo-ctrl virus is actually pTG3602 in essence. The SNP in Ixovex wasreverted back to wild type state producing the Ixo-ctrl virus. In thenumerous experiments where pTG3602 has been employed a constant lowerefficacy has been seen, as compared to the Ad5wt.

An additional advantage of Ixovex in comparison to patented adenovirusvectors of similar approach is that the Onyx-015 (Heise,Sampson-Johannes et al. 1997), -051 and -053 (Shen, Kitzes et al. 2001)all are missing the E3B gene region of the virus. This region wasoriginally deleted to enhance the safety profile of Onyx-015. It waslater found that the elimination of this region made the vectorprematurely cleared from the tumour by the immune defence (Wang, Halldenet al. 2003).

Through the western blot analysis on infections in H1299 cells it wasshown that Ixovex replicates and expresses viral proteins to the samelevel as Ad5wt. The only difference between the viruses was seen whenusing a specific antibody for the N-terminal region of E1B-55k(mouse-m2A6), a drastic increase in the E1B-156R spliceoform of theE1B-55k protein (FIG. 10). We decided to perform a number ofcomplementation experiments to verify whether indeed an increase inE1B-156R could be responsible for the increase in Oncolytic Index (OI).In FIGS. 11A, B and C and FIG. 12, we show that adenovirus type 5E1B-156R is a potent enhancer of the OI in the subfamily group C. TheE1B-156R equivalent from Ad2wt was also shown to have a positive effecton the OI of Ad5wt. Interestingly, adding Ad5-156R to Ad5wt-infectedH460 cells increased the replication of the virus, which was in linewith the much higher replication level of Ixovex as compared to theONYX-015 virus (lacking the E1B-156R gene) in H460 cells (see FIG. 6).

It is important to note that these experiments, where E1B-156R issupplemented to the virus-infected cells does not completely resembleinfection with Ixovex or another engineered virus that expressesE1B-156R. For example, during viral infection with Ixovex Ad5-156Rlevels are increased when the virus replicates, i.e. the amount ofexpression template (viral DNA) increases. In contrast, in thecomplementation experiments the E1B-156R is provided at a constanttemplate level, i.e. as the cells continue to divide during the earlyphase of the infection the plasmid harbouring the E1B-156R gene isdiluted. Thus, when the virus starts replicating E1B-156R expressionwill not increase exponentially (as would be the case for a viral copy).However, these experiments clearly show that addition of E1B-156R hasthe effect of increasing oncolytic index and suggest that E1B-156R isresponsible for this effect.

REFERENCES

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1. A recombinant adenovirus in which the proportion of the E1B-156Risoform is increased relative to wild-type levels, wherein theadenovirus has an oncolytic effect in a cancer cell.
 2. A recombinantadenovirus according to claim 1, that carries a mutation such that theproportion of the E1B-156R isoform is increased relative to wild-typelevels.
 3. A recombinant adenovirus according to claim 1 or claim 2,wherein the mutation is in the sequence of the E1B gene of theadenovirus.
 4. The recombinant adenovirus of any one of claims 1-3,where the E1B gene is mutated in one or more of the splicing recognitionregions comprising: a) the splice donor site 1 (SD1) that has thesequence GTGGC at position 2251-2255 of the Ad5 genome (position2256-2260 in Ad5 genome accession number AC_(—)000008.1 (SEQ ID NO: 41)and position 543-547 in the E1B gene (SEQ ID NO: 1)); b) the E1B-93Rsplice acceptor (SA1) that has the sequence AACAG at position 3218-3222of the Ad5 genome (position 3213-3217 in Ad5 genome accession numberAC_(—)000008.1 (SEQ ID NO: 41) and position 1500-1504 in the E1B gene(SEQ ID NO: 1)); c) the E1B-156R splice acceptor (SA2) that has thesequence TTGAG at position 3276-3280 of the Ad5 genome (position3271-3275 in Ad5 genome accession number AC_(—)000008.1 (SEQ ID NO: 41)and position 1558-1562 in the E1B gene (SEQ ID NO: 1)); d) the E1B-84Rsplice acceptor (SA3) that has the sequence TGCAG at position 3595-3599of the Ad5 genome (position 3590-3594 in Ad5 genome accession numberAC_(—)000008.1 (SEQ ID NO: 41) and position 1877-1881 in the E1B gene(SEQ ID NO: 1)); and/or e) the splice donor site 2 (SD2) that has thesequence GTACT at position 3506-3510 of the Ad5 genome (position3511-3515 in Ad5 genome accession number AC_(—)000008.1 (SEQ ID NO: 41))and position 1798-1802 in the E1B gene (SEQ ID NO: 1)).
 5. Therecombinant adenovirus of any one of claims 1-4, where the E1B genesplicing recognition regions are mutated at one or more of the followingpositions: a) nucleotide 3216 of the adenovirus Ad5 genome (accessionnumber AC_(—)000008.1) (SEQ ID NO: 41) (position 1503 in the E1B gene(SEQ ID NO: 1)); b) nucleotide 3218 of the adenovirus Ad5 genome(accession number AC_(—)000008.1) (SEQ ID NO: 41) (position 1505 in theE1B gene (SEQ ID NO: 1)); and/or c) nucleotide 3221 of the adenovirusAd5 genome (accession number AC_(—)000008.1) (SEQ ID NO: 41) (position1508 in the E1B gene (SEQ ID NO: 1)).
 6. The recombinant adenovirus ofany one of claims 1-5, where the mutation: a) removes a splice site bychanging the polynucleotide and polypeptide sequence of the E1B gene; orb) removes a splice site by changing the polynucleotide sequence of theE1B gene and retains the original polypeptide sequence.
 7. Therecombinant adenovirus of any preceding claim, where the E1B genecontains one or more of the following mutations: a) A3216G in theadenovirus Ad5 genome (accession number AC_(—)000008.1) (SEQ ID NO: 41)(position 1503 in the E1B gene (SEQ ID NO: 1)); b) G3218A in theadenovirus Ad5 genome (accession number AC_(—)000008.1) (SEQ ID NO: 41)(position 1505 in the E1B gene (SEQ ID NO: 1)); and/or c) G3221A in theadenovirus Ad5 genome (accession number AC_(—)000008.1) (SEQ ID NO: 41)(position 1508 in the E1B gene (SEQ ID NO: 1)).
 8. The recombinantadenovirus of any preceding claim, where the adenovirus is adenovirusserotype Ad5.
 9. The recombinant adenovirus of any preceding claim,where the adenovirus is adenovirus serotype Ad5 strain pTG3602.
 10. Therecombinant adenovirus of any preceding claim, where the E1B gene hasthe polynucleotide sequence according to SEQ ID NO:
 1. 11. Therecombinant adenovirus of any preceding claim, where the proportion ofthe E1B-156R isoform is increased relative to the E1B-496R isoform. 12.The recombinant adenovirus of any preceding claim, where the proportionof the E1B-496R isoform is decreased relative to wild-type levels. 13.The recombinant adenovirus of any preceding claim, where the proportionof the E1B-156R isoform is increased relative to the E1B-93R isoform.14. The recombinant adenovirus of any preceding claim, where theproportion of the E1B-156R isoform is increased relative to the E1B-84Risoform.
 15. The recombinant adenovirus of any proceeding claim, wherethe proportion of the E1B-156R isoform is increased at least 2-fold,4-fold, 10-fold, 100-fold, 1,000-fold or 10,000-fold.
 16. Therecombinant adenovirus of any proceeding claim, where the proportion ofthe E1B-496R isoform is decreased at least 2-fold, 4-fold, 10-fold,100-fold, 1,000-fold or 10,000-fold.
 17. The recombinant adenovirus ofany preceding claim, where the proportion of the E1B-156R, E1B-496R,E1B-93R, or E1B-84R isoforms refers to: a) the level of the isoformprotein that is expressed; and/or b) the level of the isoform mRNA thatis transcribed.
 18. The recombinant adenovirus of any preceding claim,where the E1B-156R isoform has a polynucleotide sequence that has atleast 80% sequence identity to SEQ ID NO:
 2. 19. The recombinantadenovirus of any preceding claim, where the E1B-156R isoform has apolypeptide sequence that has at least 80% sequence identity to SEQ IDNO:
 3. 20. The recombinant adenovirus of any preceding claim, where theE1B-496R isoform has a polynucleotide sequence that has at least 80%sequence identity to SEQ ID NO:
 4. 21. The recombinant adenovirus of anypreceding claim, where the E1B-496R isoform has a polypeptide sequencethat has at least 80% sequence identity to SEQ ID NO:
 5. 22. Therecombinant adenovirus of any preceding claim, where the E1B-93R isoformhas a polynucleotide sequence that has at least 80% sequence identity toSEQ ID NO:
 6. 23. The recombinant adenovirus of any preceding claim,where the E1B-93R isoform has a polypeptide sequence that has at least80% sequence identity to SEQ ID NO:
 7. 24. The recombinant adenovirus ofany preceding claim, where the E1B-84R isoform has a polynucleotidesequence that has at least 80% sequence identity to SEQ ID NO:
 8. 25.The recombinant adenovirus of any preceding claim, where the E1B-84Risoform has a polypeptide sequence that has at least 80% sequenceidentity to SEQ ID NO:
 9. 26. A recombinant adenovirus of any precedingclaim, where the cancer cell is a neoplastic cell that substantiallylacks p53 function.
 27. A recombinant adenovirus of any preceding claim,where the oncolytic effect comprises: a) viral infection of cells; b)selective replication of the viral genome in (p53-deficient) cancercells leading to preferential virus-mediated cell lysis in(p53-deficient) cancer cells, and the release of viral particles forfurther infection events.
 28. A polynucleotide encoding the recombinantadenovirus of any preceding claim, which is optionally a vector suitablefor adenovirus production in a host cell.
 29. A host cell comprising apolynucleotide encoding the recombinant adenovirus of any precedingclaim.
 30. A method of treating cancer, characterized by neoplasticcells that substantially lack p53 function, in a patient in need oftreatment, comprising: a) administering a dose of the recombinantadenovirus of any preceding claim; b) allowing sufficient time for saidrecombinant adenovirus to infect neoplastic cells of said cancer; and c)optionally administering further doses of the recombinant adenovirus.31. A composition comprising a recombinant adenovirus of any precedingclaim for use as a therapeutic agent.
 32. A composition comprising arecombinant adenovirus of any preceding claim for use in treating apatient with cancer, characterised by neoplastic cells thatsubstantially lack p53 function.
 33. A method of rendering an adenovirusoncolytic by modifying the sequence of the E1B gene to increase thelevel of the E1B-156R isoform relative to the level in the equivalentwild-type adenovirus.